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TRANSCRIPT
PERPUSTAKAAN UMP
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DETERMINATION OF GLUFOSINATE-AMMONIUM AND MALATHION IN RESIDUE OIL FROM PALM PRESSED FIBER USING HIGH PERFORMANCE
LIQUID CHROMATOGRAPHY
MUHAMMAD HAZIQ BIN RUSSLI
Report submitted in partial fulfillment of the requirements for the award of Bachelor of Applied Science (Honour) in Industrial Chemistry
Faculty of Industrial Sciences & Technology UNIVERSITI MALAYSIA PAHANG
2012
ABSTRACT
DETERMINATION OF GLUFOSINATE AMMONIUM AND MALATHION IN RESIDUE OIL FROM PALM PRESSED FIBER USING HIGH PERFORMANCE LIQUID CHROMATOGRAPHY
A study on the determination of organophosphorus pesticides (OPPs), namely glufosinate-ammonium and malathion in residue from palm pressed fiber (PPF). Three PPF samples were taken from three different areas, FELDA Lepar, FELDA Chini 1 and FELDA Chini 2.The extraction of oil residue in PPF was carried out using supercritical fluid extraction (SFE), using supercritical carbon dioxide (CO2) at 3000 psi in isothermal in 60°C for 60 minutes. Residue then were diluted uisng acetonitrile. Chromatogram of peak analyte was determine using Waters Alliance high performance liquid chromatography (HPLC) Dissolution System Series liquid chromatography interfaced to a 2998 Photodiode Array Detector. Determination was first done using standard malathion and glufosinate-ammonium of 10, 15 and 20 ppm run on a iscoratic elution mode for 30 minutes. Optimization method later was done using water and acetonitrile as mobile phase and run for 16 minutes for malathion and 20 minutes for glufosinate-ammonium. Malathion peak is retained at 8.0 minutes while glufosinate-ammonium at 16.0 minutes. Calibration curve of the standards were plot with R2 values of 0.9085 for malathion and 0.9249 for glufosinate-ammonium. The detection limits (LOD) were 10.3 mg/L for malathion and 14.1 mg/L for glufosinate-ammonium. Later, determination of malathion and glufosinate-ammonium were done on real samples. For malathion determination, sample from Lepar showed to have concentraion of 19.46 ppm, Chini 1 (14.34 ppm) and Chini 2 (9.72 ppm). Determination of glufosinate-ammonium showed sample from Lepar to have concentraiton of 12.64 ppm, Chini 1 (17.72 ppm) and Chini 2 (39.64 ppm).
V
Satu kajian untuk menentukan kehadiran racun perosak jenis organophosphorus (OPPs), iaitu glufosinate-ammonium dan malathion di dalam mendakan minyak daripada fiber kelapa sawit tertekan (PPF) dijalankan. Tiga PPF sampel diambil dari tiga tempat berlainan, FELDA Lepar, FELDA Chini 1 dan FELDA Chini 2. Proses pengekstrakan mendakan minyak dilakukan dengan menggunakan supercritical fluid extraction (SFE), dengan menggunakan supercritical karbon
dioksida (CO2) pada tekanan 3000 psi didalam suhu tetap 60°C selama 60 minit. Mendakan minyak kemudian dilarutkan menggunakan acetonitirle. Kromatogram puncak komponen sasaran dikenal pasti dengan menggunakan Waters Alliance high performance liquid chromatography (HPLC) Dissolution System Series liquid chromatography yang disambungkan kepada 2998 Photodiode Array Detector. Proses dimulakan dengan menggunakan sampel malathion dan glufosinate standard berkepekatan 10, 15 dan 20 ppm, pada mod aliran isokratik selama 30 minit untuk mendapatkan proses yang optimum. Kemudian proses optimum dijalankan dengan menggunakan air dan acetonitrile sebagai mobile phase selam 16 minit untuk malathion dan 20 minit untuk glufosinate-ammonium. Puncak malathion dikenal pasti muncul pada minit ke 8 dan glufosinate-ammonium pada minit ke 16. Lengkungan penentu ukuran kemudian diplotkan dengan nilai R2
adalah 0.9085 untuk malathion dan 0.9249 untuk glufosinate-ammonium. Had penentuan (LOD) pula adalah 10.3 mg/L untuk malathion dan 14.1 mg/L untuk glufosinate-ammonium. Kemudian, kehadiran malathion dan glufosniate-ammonium dieknal pasti pada sampel sebenar. Unutk malathion, sampel Lepar mencatatkan kepekatan 19.46 ppm, Chini 1 (14.34 ppm) dan Chini 2 (9.72 ppm). Untuk glufosinate-ammonium pula, sampel dari Lepar mencatatkan kepekatan 12.64 ppm, Chini 1(17.72 ppm) dan Chini 2 (39.64 ppm).
vi
TABLE OF CONTENTS
Page
SUPERVISOR'S DECLARATION II
STUDENT'S DECLARATION
ACKNOWLEDGEMENTS 1V
ABSTRACT V
ABSTRAK
TABLE OF CONTENTS
LIST OF TABLES ix
LIST OF FIGURES xi
LIST OF ABBREVIATIONS xiii
CHAPTER 1 INTRODUCTION
1.1 Background of Study 1
1.2 Objectives 2
1.3 Problem statement 3
1.4 Scopes of Study 3
CHAPTER 2 LITERATURE REVIEW
2.1 Introduction to the palm oil 4
2.1.1 Oil palm 5
2.2 Organophosphates 6
2.2.1 Organophosphorus pesticides 6
2.2.2 Glufosinate 7
2.2.3 Malathion 9
2.3 Supercritical Fluid Extraction 10
2.4 High Performance Liquid Chromatography 12
2.4.1 Introduction 12
2.4.2 Instrumentation 13
VII
WE
CHAPTER 3 METHODOLOGY
3.1 Introduction 15
3.2 Experimental design 16
3.2.1 'Reagent and materials 16 3.2.2 Instrumentations 16 3.2.3 Sample treatment 17
3.4 Flow chart of study 20
CHAPTER 4 RESULTS AND DISCUSSION
4.1 HPLC parameter optimization 21
4. 1.1 Malathion 23 4.1.2 Glufosinate-ammonium 26
4.2 Method validation 29
4.2.1 Limit if detection 29 4.2.2 Limit of quantification 30
CHAPTER 5 CONCLUSIONS & RECOMMENDATIONS
5.1 Conclusions 31
REFERENCES
LIST OF TABLES
Table No. rage
3.1 HPLC solvent system for malathion analysis 17
3.2 HPLC solvent system for glufosinate-ammonium analysis 17
4.1 Isocratic mode 22
4.2 Gradient elution (malathion) 22
4.3 Gradient elution (glufosinate-ammonium) 22
4.4 Data for malathion at different sampling area 25
4.5 Data for glufosinate-ammonium at different sampling 28
area
4.6 Data for LOD value for malathion and glufosinate- 29
ammonium
4.7 Data for LOQ value for malathion and glufosinate- 30 ammonium
ix
LIST OF FIGURES
Figure No.Page
2.1 Parts in palm oil fruit 5
General chemical structure of OPPs with R' and R2 6 2.2
as alkyl substitutions
2.3 Chemical structure of glufosinate ammonium 8
2.4 Chemical structure of malathion 9
2.5 Typical pressure-temperature projection of a phase diagram of pure material 11
3.1 Dilution of pure stock to 1000 ppm standard solution 18
3.2 Standard is homogenized by vortex mixture 18
3.3 Flow chart of study 19
4.1 Chromatogram of malathion (A) 10 ppm 22
(B) 15 ppm 23
(C) 25 ppm 23
4.2 Calibration curve for malathion 24
4.3 Chromatogram of glufosinate-ammonium (A) 10 ppm 25
(B) 15 ppm 26
(C) 20 ppm 26
4.4 Calibration curve for glufosinate-ammonium 27
X
LIST OF ABBREVIATIONS
AChE AcetylcholineSteraSe
CNS Central nervous system
CO2 Carbon dioxide
co Crude palm oil
EPA Environmental Protection Agency
GC Gas chromatography
HPLC High performance liquid chromatography
LC Liquid chromatography
LSC Liquid solid chromatography
NMR Nuclear magnetic resonance
OPPs Organophosphorus pesticides
PDA Photodiode array detector
PPF Palm pressed fiber
SFE Supercritical fluid extraction
UV Ultraviolet
Xi
CHAPTER 1
INTRODUCTION
1.1 BACKGROUND OF STUDY
Malaysia is the world's largest exporter of palm oil, accounting for
about 61.1% or 10.62 million tons of the total exports of 17.37 million tons
based on 2001 statistics (Hai, T.C., 2002). In the early 1970s 56,674 hectares
of land was planted with oil palm, increased to the latest data at 2002, about
3.6 million hectares are used to plant oil palm trees (Abdullah, R., 2003; Teoh,
C.H., 2002). The increasing areas of plantation plant contribute to the
increasing requirement of pesticides. Wibawa, W. et al. stated that in 2004, the
herbicide usage in Malaysia was 67.49% of the total pesticides used and in
2005, more than 15.6 million liter was used in oil palm alone.
Pesticides consists of insecticide, fungicide, herbicide,
organophosphorus pesticides (OPPs). Herbicides are known to increase
specific plant disesase (Altman and Campbell, 1977; Hornby et al., 1998;
Mekwatanakarn and Sivasithamparam, 1987), and several are reported to
influence micronutrient availability (Evans et al., 2007; Huber et al., 2004,
2005). Some 1000-1500 new chemicals are manufactured each year with
perhaps 60,000 chemicals in daily use (OECD, 1981). Glyphosate and
glufosinate are two of the world's most widely used non-selective OPPs
herbicides. Although they are not considered to present a major risk to human
health, recent evidence suggest that exposure may cause neurological disorders
1
(BarboSa, E.R., et al., 2001). Glufosinate can affect the nervous system.
Glutamate IS an 'excitatory' neurotransmitter in the brain, and it appears to
affect some of the processes in the nervous system that normally involve
glutamate.
The usage of pesticide is to eliminate the non-desired plants, thus
stimulate the growth of the palm oil plant, can be resulting of the presence of
pesticide in the plant, or in residue oil. The residue oil is in the palm pressed
fiber (PPF), a byproduct of palm oil milling. It usually used to generate
electricity supply for mill and small housing estates around the mill (Basiroh
and Simeh, 2005). Malaysia produce about 8.56 million tons of PPF per year
(Hussain et al., 2003) at about 5-6% residue oil content, it such a waste to
burnt it to produce electricity. Some of the contents in the residue oil are
highly valuable which are carotenes, tocols, sterols, squalene and
phospholipids.
Many attempts have been made to recover the residue oil from PPF
including solvent extraction and supercritical fluid extraction (SFE) (Choo, et
al., 1996; Lau, et al., 2006;de Franca and Meireles, 2000). Application of
supercritical carbon dioxide (SC-0O 2) extraction has advantages over the
solvent extraction method as it uses non-hazardous and non-inflammable CO2
when compared to highly flammable petroleum-based solvent such as hexane
or acetone (Lau, H.L.N., et al., 2007). Sc-c0 2 extraction has been used to
concentrate minor constituents from various oilseed and other products. These
include the isolation of tocopherols from soybean and canola oil deodorizer
distillates (Mendes, et al., 2002), sterols and tocopherols from olive oil
(Ibanez, et al., 2002), squalene from PPF and palm leaves (Britigh, et al., 1995;
Choo, et al., 1996; de Franca, et al., 2000).
2
1.2 OBJECTIVE
The objective of this study is:
1. To analysis the presence of OPPs in real sample from different
area.
2. To conduct real sample analysis of OPPs using supercritical fluid
extraction (SFE) using supercritical CO2.
3. To optimize the parameter (mobile phase) for the determination of
OPPs using high performance liquid chromatography (HPLC).
1.3 PROBLEM STATEMENT
Previous experiments use Soxhlet extraction, which is a versatile
classical sample preparation technique prescribed in many standard analytical
methods. Soxhlet extraction is considered to be a time consuming, tedious and
uses a lot of extraction solvent.
1.4 SCOPE OF STUDY
Scope of study focus on determination of glufosinate ammonium and
malathion in residue oil from palm pressed fiber (PPF). In this study, samples
from different area was used to compare the presence of selected OPPs.
Samples were extracted using SFE by supercritical carbon dioxide (CO2).
Extracted analytes then were analyzed using high performance liquid
chromatography (HPLC). Optimization of extraction parameter will not be
carried out since this study is more towards to environmental analysis.
3
CHAPTER 2
LITERATURE REVIEW
2.1 INTRODUCTION TO THE PALM OIL
Oil palm refers to the palm, Elais guineensis Jacq. (Hatrley, 1988). Oil
palm produces two types of oil, palm oil from the fibrous mesocarp and palm
kernel oil, a lauric oil from the palm kernel. The development of the oil palm
industry in Malaysia is attributed to Frechman, Henri Fauconnier and his
association with Hallet. In 1911, Fauconnier visited Hallet's oil palm
development in Sumatra and had purchased some oil palm seeds and these
were planted at his Rantau Panjang Estate in Selangor (Tate, 1996). With
seedlings obtained from the 1911 and 1912 importation, Fauconnier
established the first commercial oil palm planting at Tennamaram Estate, to
replace an unsuccessful planting of coffee bushes (Tate, 1996).
Gray (1969) and Hacharan Singh (1976) had analyses of palm oil
industry in Malaysia, and classified the development of the industry in
Peninsular Malaysia into three distinct phases. Starting with the experimental
phase for the late 1800s early 1900 to 1916 while the plantation development
phase commenced in 1917 with Tennamaram Estate until about 1960. To
reduce the dependence of the national economy on natural rubber, the
Government decides to expand on oil palm, by the recommendation of the
World Bank Mission in 1955. The palm oil industry has since undergone two
further phases, from 1970 with the expansion of large scale planting in Sabah
4
ARP1<
ARP ) I 1W ARPd
and Sarawak and from around 1955 when Malaysian extended their upstream
operations off-shore, particularly to Indonesia where there is adequate supply
of workers and availability of land for plantation development and cost of
production is lower than in Malaysia.
2.1.1 Oil Palm
An oil palm fruit bunch consists of two main parts, the stalk and the
fruit lets. The fruit lets are made up of pericarp (mesocarp and endocarp) and
the nut. Two types of oils are produced from the fruit, namely palm oil from
the oil cells in mesocarp and palm kernel oil from seed (kernel) of the nut.
These oils are extracted and recovered separately in palm oil mill and kernel
crushing plant. In general a palm oil mills produce the following products and
by-products from the fresh fruit bunch: 20% crude palm oil (CPO), 23% empty
bunch, 5-7% kernel depending on type of fruit and 15% fiber. After CPO is
extracted from the fruit by screw press, this fiber is referred to as palm pressed
fiber (PPF) and contains 5-6% residue oil, which is burnt, along the with the
valuable oil to provide steam and power for the mill. PPF is highly valuable
with high level of phytonutrients content like carotenoids, tocopherols and
tocotrienol. The extraction can be done using hexane of food grade as solvent.
5
Figure 2.1: Parts in palm oil fruit.
Source: http://www.etawau.com
2.2 ORGANOPHOSPHATES
2.2.1 Organoph0sPh0s Pesticides
OPPs are normally esters, amides or thiol derivatives of phosphoric,
phosphonic, phosphorothioic or phosphonothioic acids. Most are only slightly
soluble in water and have a high oil-to-water partition coefficient and low
vapor pressure (International Programme on Chemical Safety Databank,
1986).
K' 0 or 5)
R27
Figure 2.2: General chemical structure of OPPs with R' and R 2 as alkyl substitutions.
Source: International Programme on Chemical Safety Databank. 1986.
R' and R2 are usually simple alkyl or alkyl groups, both of which may
be bonded directly to phosphorus (in phosphinates), or linked via —0-, or —5-
(in phosphates), or R' may be bonded directly and R 2, bonded via one of the
above groups (phosphonates). In phosphoramidates, carbon is linked to
phosphorus through an —NH group. The group X can be any one of a wide
variety of substituted and branched aliphatic, aromatic, or heterocyclic groups
linked to phosphorus via bond of some liability (usually —0- or —5-) and is
referred to as the leaving group. The double-bonded atom may be oxygen or
sulfur and related compounds would, for example be called phosphates or
Phosphorothioates (the nomenclature 'thiophosphate' or 'thionophosphate' is
now less used).
6
7
In 1820, effects on human beings from the exposure to the OPPs can
cause musacariflic, nicotinic and central nervous system (CNS) manifestations.
Symptoms may develop rapidly or there may be a delay of several hours after
exposure before they become evident. The delay tends to be a longer in the
case of more lipophilic compounds, which also require metabolic activation.
Symptoms may increase in severity for more than one day and may last for
several days.
Continuous long-term exposure to high levels of OPPs may precipitate
typical cholinergic symptoms, though most of the compounds do not
accumulate extensively in the body.
As OPPs are commonly used on vegetation in Malaysia, such as
acephate, diazinon, dichiorvos, dimethoate, fenitrothion, malathion,
metham idophos, phenoate, prothiophos, triaziphos and tolcofos-methyl,
numerous methods have been developed for the analysis of OPPs (Lee et al.,
1991). Bio-assay, enzymatic methods, nuclear magnetic resonance (NMR),
mass spectrometry, polarography and partition function are among applied
technique for analysis of organophosphorus compounds (Thomas, 1974).
2.2.2 Glufosinate
The ammonium salt of glufosinate was first registered in the U.S. for use
as an herbicide in 1993 by Hoescht Celanese (Caroline, C., 1996). Glufosinate
inhibits the activity of an enzyme involved in the synthesis of the amino acid
glutamine. This enzyme is called glutamine synthethase. Essentially, glufosinate
acts enough like glutamate, the molecule used by this enzyme to make glutamine,
that it blocks the enzyme's usual function. Glutamine synthetase is also involved
in ammonia detoxification. Treatment with glufosinate leads to reduced
glutamine and increased ammonia levels in plant tissues. This causes
Photosynthesis to stop, the plant to wither, and then die within a few days
8
(l-1orleifl G., 1994). Although most herbicides are not nerve poisons, glufosinate
can affect the nervous system. Glutamate is an 'excitatory' neurotransmitter in
the brain, and it appears to affect some of the processes in the nervous system
that normally involve glutamate (U.S. EPA, 1985).
Whether or not glufosinate causes cancer is ambiguous. One of the
studies submitted in support of its registration, a long-term feeding study in rats,
showed an increase in the frequency of adrenal medullary tumors. (U.S. EPA,
1992). But still, the lack of publicly available studies about the ability of
g1ufosinatecOfltainiflg products to cause cancer cannot confirm this study.
There are some cases that glufosinate was found in the edible parts of
spinach, radishes, wheat, and carrots that had been planted 120 days after
treatment with a glufosinate-containing herbicides (U.S. EPA, 1988).
0
110
H3COH
ONH4i- 2
Figure 2.3: Chemical structure of glufosinate ammonium
Source: Journal of Pesticide Reform, 2003
9
IUPAC name : Ammonium (2R5)-2-amino-4-
(methylphosphinato)butyric acid
GAS number : 77182-822
Molecular weight
Molecular formula
Log Kow
Boiling point
Melting point
Vapor pressure
Water solubility at 25°C
198.16 gmol'
C5H15N204P
-5.34
433.91°C
272.12°C
1.58 e' mmHg at 25°C
1.0 e6 mg/L
2.2.3 Malathion
Malathion is an insecticide in the organophosphate chemical family. It
is one of the oldest insecticides in that family and has been used since 1950
(Ware, G.W., 2000). Malathion is the most commonly used insecticide in the
U.S., the U.S. Environmental Protection Agency (EPA) estimates that annual
use of malathion is over 30 million pounds (Donaldson, D.T., and Grube, A.,
2002). Malathion kills insects by converting inside animals into maloxon, a
chemical relative that inhibits an important central nervous system enzyme
called acetylcholinesterase (AChE) (U.S. EPA, 2000). AChE is involved with
the transmission of nerve impulses. When this enzyme is inhibited, the
transmission 'jams' resulting in restlessness, hyperexcitability, convulsions,
paralysis, and death. All insecticides in the OPPs chemical family share a
similar mode of action (Journal of Pesticide Reform, 2003).
10
Figure 2.4: Chemical structure of malathion
Source: Journal of Pesticide Reform, 2003
IUPAC name
CAS number
Molecular weight
Molecular formula
Log Kow
Boiling point
Melting point
Vapor pressure
Water solubility at 25°C
Diethyl
[(dim ethoxyphosphoroth ioyl)sul fanyl] butanedioate
121-75-5
330.35 g mo!1
C10H9N204P
2.29
433.91'C
:272.12°C
1.58 e' mmHg at 25°C
1.Oe6mg/L
2.3 SUPERCRITICAL FLUID EXTRACTION
The unique solvent properties of supercritical fluids were first reported
well over 100 years ago in 1879 by Hannay and Hogarth (Jonin, T.M., et al.,
1986), who measured the solubility of several inorganic salts in supercritical
ethanol. By 1980s and 1990s, supercritical fluids have been used in several
I ndustrial processes, including decaffeination of coffee (Brunner, G., et al., 2005)
and tea, extraction of hop flavor for the beer industry and extraction of lipids and
11
aromas from plant material. Other use including as solvents for supercritical
chromatography (Jonin, T.M., et al., 1986).
A fluid is considered supercritical if it exists at conditions above its
critical pressure and temperature. These critical values correspond to conditions
in which condensation into a liquid or evaporation into a gas is no longer
possible.
Figure 2.5 : Typical pressure-temperature projection of a phase diagram for pure
material
Source : http://www.eolss.net/Sample-Chapters/C I 01E5- 1 O-04-08.pdf
The supercritical fluids most commonly used are CO 2, ethane, ethane,
propane, ammonia and water. However, CO 2 is preferred because of its
convenient critical temperature, cost, chemical stability, non-flammability and
non-toxicity. Disposal of CO 2 is more environmentally friendly than for most
other organic solvents typically used in extraction processes. It can be obtained
in large quantities as a byproduct of several reactions, such as fermentation,
combustion and ammonia synthesis. Another advantages of using supercritical CO2 is that once the extract returns to standard conditions of pressure and
12
temperature, the CO2 returns to a gas phase and the extracted product precipitates
since it is no longer soluble in the gas (Jonin, T.M., et al., 1986).
Recently, a different approach has been used to increase the extraction
efficiency using pure CO 2 . That is, raising the extraction temperature while using
pure CO2 . At higher pressure and temperature, the solvent strength of the carbon
dioxide is increased. This helps to increase the extraction recovery. However,
when extraction temperatures are in the neighborhood of the vapor pressure of
the compound of interest, the extraction recovery is significantly increased. In
other word, the solubility of the analyte is influenced not only by the density of
the CO2 , but also by the vapor pressure of the target analyte. And solubility of
the compound of interest is not significantly influenced by the density of CO2,
compared to the vapor pressure of the analyte (Patel, S., 1999).
2.4 HIGH PERFORMANCE LIQUID CHROMATOGRAPHY
2.4.1 Introduction
High performance liquid chromatography (HPLC) is a technique that
has arisen from the application to liquid chromatography (LC) of theories and
instrumentation that were originally developed for gas chromatography (GC)
(Sandie, L., and John, B., 1987). In the original method, an adsorbent for
instance alumina or silica is packed into a column and is eluted with a suitable
liquid. The separation of the solutes is possible if there are differences in their
adsorption by the solid. This method is called adsorption chromatography or
liquid solid chromatography (LSC) (Sandie, L., and John, B., 1987).
The efficiency could be improve if the particle size of stationary phase
materials used in LC could be reduced. As HPLC has developed, the particle
Size of the stationary phase materials used in LC has become progressively smaller. The stationary phases used today are called microparticulate column
packings and are commonly uniform, porous silica particles, with spherical or
irregular shape, and nominal diameters of 10, 5 or 3 J.tm.
2.4.2 Instrumentation
There are five major HPLC components and their functions. It consists
of pump, injector, column, detector and computer. A typical HPLC set-up uses
an isocratic pump, a water/buffer and methanol eluent, a C 18 column and a
ultraviolet (UV) -detector. Injections in the range 5-100 j.tL is performed by an
autoinjector and the peak area response is evaluated by an integrator.
Basically, a pump is used to propel the solvent or eluent. It considered
the most important component in LC system and its basic parameter is
pumping system. The systems are positive-pressure system or constant-
pressure pump and positive-flow system or constant-flow pump (Yost, R.W. et
al., 1980).
The primary advantages of most constant-pressure pumps are
simplicity and freedom from pulsations, resulting in smooth baselines. But it
also suffers from several disadvantages. Flow rates can be change if the
solvent viscosity changes due to temperature change. This translates to
component location and identification becomes inaccurate or impossible. In
quantitative analysis, the UV and the refractive index detectors used most
frequently in LC are concentration sensitive. Changes in flow result in changes
the eluent/sample dilution ration, or in other words changes in concentration
which show up as changes in peak area.
While the constant-flow systems are generally of two basic types:
reciprocating and positive displacement (syringe) pumps. The basic
advantages of such system are their inherent ability to repeat elution volume
13
and area, regardless of viscosity changes or column blockage or settling
occurrence, up to the pressure limit of the pump (Yost, R.W. et al., 1980).
It is obvious that these pumps deliver a series of 'pulses' of the mobile
phase. Detector will be disturbed by the pulsations, especially at high
sensitivities. To avoid this problem, several methods have been developed and
most simple involves placing a large (often 50 feet) coil of narrow-bore tubing
in the line between the pump and the column. It acts as absorbent, absorbing
the energy of the pulsations as the pump strokes.
The primary consideration in injector design is the need to provide a
low-volume, completely swept area to avoid sample diffusion and exponential
dilution (Yost, R.W., et al., 1980). There are two systems, injection through a
septum which is historically been the most frequently used and septumless
syringe-injector valves.
Ideally, the septum injection is designed so it can be swept into the
column without back-mixing and subsequent band spreading. Its design so that
when the needle to close more rapidly, effectively sealing the system. When
this injector is used, 50-60 injections are routinely made into ordinary silicone
rubber septurns, often at pressures as high as 2000 lb/sq.in . Most syringe used
has the capacity of 10 microliters. Needle length or injection depth must be
regulated according to the instrument since inserting the needle into the top of
the column usually results in a plugged syringe and no injection of sample
(Yost, R.W., et al., 1980).
14
CHAPTER 3
METHODOLOGY
3.1 INTRODUCTION
To achieve the objective of this study, various literature reviews had
been done to improve the understanding of this study. Through the readings,
there are many methodologies to be reconsidered, depend on the researcher
capability to reproduce and the availability of such as reagents, materials and
instruments. The result from this study cannot be the sole measurement of the
achievement of this study though, as the result depends on various factors,
considering some of its are out of researcher control.
From the literature reviews, the methodology of this study can be
divided into two stages. The first stage is optimization of parameters and
second stage is real sample analysis. The outcome result from the real sample
analysis may not as it expected from the first stage. This may be due to
different behavior of real sample form the standard sample when analysis takes
place.
15
3.2 EXPERIMENTAL DESIGN
3.2.1 Reagent.and Materials
Materials
Fresh palm pressed fiber (PPF) collected from a local palm oil mill
(FELDA, Pahang, Malaysia). Freshly collected samples were dried at 60-70°C
in oven for about 12 hours. The samples is checked and mix homogenously for
every an hour to prevent samples burnt. The average water content before
drying was 17%.
Reagents and solutions
Glufosinate-ammonium, and malathion analytical standards were
purchased from Sigma Aldrich (Germany), methanol, acetonitrile, acetic acid,
ammonia, water, and deionized water.
3.2.2 Instrumentations
HPLC analyses were run on a Waters Alliance HPLC Dissolution
System series liquid chromatpgraphy (Waters Corporation, Milford,
Massachusetts, USA) interfaced to a 2998 Photodiode Array Detector (PDA).
The analytical column was packed with C 18 stationary phase (150 mm x 4.6
mm l.D., 4.5 gm). The detection wavelength of the detector was set at 254 nm.
16